Dye Entry Research Articles

Visualization of MG53-mediated Cell Membrane Repair Using <em>in vivo</em> and <em>in vitro</em> Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using <em>in vivo</em> and <em>in vitro</em> Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Analysis of marginal adaptation and sealing to enamel and dentin of four self-adhesive resin cements

This in vitro study compared the marginal adaptation of all-ceramic MOD-inlays luted to human molars with four self-adhesive resin cements. Thirty-two human third molars were randomly assigned to four test groups (n = 8 per group). MOD cavities were prepared with approximal finishing lines in dentin and enamel. All-ceramic Empress 2 inlays were luted with four self-adhesive cements (Clearfil SA, iCEM, Bifix SE, seT). Oral stress was simulated by 90 day storage in water as well as by thermal and mechanical loading (TCML, 1.2 × 10(6) × 50 N, 6,000 × 5°/55°, 1.6 Hz). The marginal fit was evaluated by scanning electron microscopy (SEM) and dye penetration. Data were analyzed with the ANOVA/Tukey's test (α = 0.05). The SEM investigation of the gingival cement margins (cement-tooth interface) showed values of perfect margin [percent] (means ± SD) after simulated aging between 84 ± 9% and 95 ± 5% for enamel and 80 ± 9% and 92 ± 3% for dentin. In enamel, seT showed significantly higher marginal integrity than iCEM after water storage and TCML (post hoc; p = 0.011). Furthermore, the marginal adaptation of iCEM in enamel deteriorated by simulated aging (p = 0.014, ANOVA). Mean values of dye penetration (percentage of dye entry into dentin) at the investigated restorations margins ranged between 3% and 8% for enamel and 12% and 22% for dentin. Clearfil SA, iCEM, and seT showed lower dye penetration in enamel than in dentin (Clearfil SA: p = 0.013, iCEM: p = 0.044, seT: p = 0.003). The results suggest that the four self-adhesive luting agents investigated seem to successfully bond to dentin-restricted as well as to enamel-restricted cavities, predicting good clinical performance.

Recovery of mechano-electrical transduction in rat cochlear hair bundles after postnatal destruction of the stereociliar cross-links

Mechano-electrical transduction (MET) in the stereocilia of outer hair cells (OHCs) was studied in newborn Wistar rats using scanning electron microscopy to investigate the stereociliar cross-links, Nomarski laser differential interferometry to investigate stereociliar stiffness and by testing the functionality of the MET channels by recording the entry of fluorescent dye, FM1-43, into stereocilia. Preparations were taken from rats on their day of birth (P0) or 1–4 days later (P1–P4). Hair bundles developed from the base to the apex and from the inner to outer OHC rows. MET channel responses were detected in apical coil OHCs on P1. To study the possible recovery of MET after disrupting the cross-links, the same investigations were performed after the application of Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and allowing the treated samples to recover in culture medium for 0–20 h. We found that the structure and function were abolished by BAPTA. In P0–P1 samples, structural recovery was complete and the open probability of MET channels reached control values. In P3–P4 samples, complete recovery only occurred in OHCs of the outermost row. Although our results demonstrate an enormous recovery potential of OHCs in the postnatal period, the structural component restricts the potential for therapy in patients.

Squalamine, an original chemosensitizer to combat antibiotic-resistant Gram-negative bacteria

Sir, The worldwide dissemination of multidrug-resistant (MDR) pathogens has severely reduced the efficacy of our antibiotic arsenal and increased the frequency of therapeutic failure. To reduce the intracellular accumulation of antibiotics, a key bacterial response is to stringently control membrane transporters involved in the diffusion of drugs through the envelope into the cell and those involved in the expulsion of antibiotics, the so-called ‘in and out’ flux. In Gram-negative bacteria, the outer membrane is an additional barrier to antibiotic penetration. Numerous antibiotics that are active against Grampositive are much less active against Gram-negative bacteria. An attractive approach to circumvent bacterial resistance is the development of a chemosensitizer agent to promote an increase of the internal antibiotic concentration in resistant strains. This agent will be used in combination with classic antibiotics to bypass the mechanical and enzymatic barriers that reduce the intracellular concentration of the active antibacterial drug. Squalamine exhibits interesting antimicrobial activity, suggesting its potential application to combat resistant pathogens. It modifies membrane integrity by increasing permeability, as demonstrated by ATP release and dye entry in Gram-negative bacteria, and it has a moderate cytotoxicity at doses that kill MDR bacterial pathogens. The aim of this study was to evaluate the effect of subinhibitory concentrations of squalamine on the activity of various antibiotic classes against different resistant bacteria. The chemosensitizer activity of squalamine was studied by determining the MICs of various usual antibiotics in the presence of subinhibitory concentrations of squalamine. These concentrations corresponded to 1/5 and 1/10 of the respective squalamine MICs determined for each bacterial strain. A control was included by using the well-described efflux pump inhibitor phenylalanine arginine b-naphthylamide (PAbN) to evaluate the involvement of efflux. The strains were grown on Mueller–Hinton broth (Becton Dickinson) at 378C. The Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae and Pseudomonas aeruginosa strains used have been previously described. Results were read after 18 h at 378C and the MIC values are the means of three independent experiments. The results for documented E. coli strains exhibiting various porins and efflux pump profiles indicated that, even at the low subinhibitory concentration tested (MIC/10), squalamine had an effect on the activity of various different antibiotic classes (Table 1). A significant reduction in chloramphenicol, tetracycline and ciprofloxacin MICs was observed for AG100 and AG100Atet in the presence of squalamine. AG100Atet is an acrAB2 strain that overproduces several drug efflux pumps and down-regulates porin expression. An effect was also obtained on cefepime susceptibility, indicating that squalamine may favour the uptake of this b-lactam molecule via the permeabilizing effect previously reported. It is worthwhile mentioning that squalamine also increases the activity of tested antibiotics against susceptible cells, AG100 and AG100A, demonstrating that the effect is not focused on a resistance mechanism but is related to increased drug penetration. The MICs of erythromycin were not significantly modified by the addition of squalamine, indicating the prominent role of efflux mechanisms for this antibiotic group. In the K. pneumoniae KP55 clinical isolate, the MICs determined in the presence of squalamine were significantly decreased, e.g. 16–32-fold for chloramphenicol, ciprofloxacin, tetracycline and cefepime (Table 1). Squalamine was more efficient at increasing tetracycline and cefepime activity against E. aerogenes than PAbN, highlighting the role of uptake in susceptibility to the two antibiotics. The effect of squalamine on strains devoid of porin (KP63 and KP55) indicated an increase in penetration for cefepime and ciprofloxacin, which normally use porin channels to pass the membrane barrier. Regarding P. aeruginosa and E. aerogenes strains, the results similarly suggest that when the mechanical barrier, restricted uptake or efficient efflux pump participates in resistance, squalamine is able to significantly increase bacterial susceptibility. Squalamine has an effect on the susceptible strains (E. coli, E. aerogenes and P. aeruginosa) tested in this study, suggesting that squalamine may chemosensitize the membrane of nonresistant bacteria and promote the use of a decreased amount of antibiotics. We have previously demonstrated that the alteration of lipopolysaccharide involved in polymyxin-resistant clinical isolates only moderately changes squalamine susceptibility. Consequently, the use of squalamine against polymyxin-resistant isolates selected during colistin treatment, a therapy that is now proposed for the MDR phenotype, may be an attractive hypothesis for the development of future drug combinations. In the bacterial resistance context, and because of its action and its relative insensitivity to efflux resistance mechanisms, squalamine may be a fruitful partner for the development of combinations, such as b-lactams/b-lactamase inhibitors, to combat MDR pathogens. This use allows us to complete our new strategies by promoting the penetration (influx) of antibiotics, in parallel with inhibitors of efflux pumps andb-lactamase inhibitors. This aspect is especially important, since some recent molecules having an original antibacterial spectrum exhibit restricted diffusion through the Research letters

P2X antagonists inhibit styryl dye entry into hair cells

The styryl pyridinium dyes, FM1-43 and AM1-43, are fluorescent molecules that can permeate the mechanotransduction channels of hair cells, the sensory receptors of the inner ear. When these dyes are applied to hair cells, they enter the cytoplasm rapidly, resulting in a readily detectable intracellular fluorescence that is often used as a molecular indication of mechanotransduction channel activity. However, such dyes can also permeate the ATP receptor, P2X2. Therefore, we explored the contribution of P2X receptors to the loading of hair cells with AM1-43. The chick inner ear was found to express P2X receptors and to release ATP, similar to the inner ear of mammals, allowing for the endogenous stimulation of P2X receptors. The involvement of these receptors was evaluated pharmacologically, by exposing the sensory epithelium of the chick inner ear to 5 μM AM1-43 under different experimental conditions and measuring the fluorescence in hair cells after fixation of the tissue. Pre-exposure of the tissue to 5 mM EGTA for 15 min, which should eliminate most of the gating “tip links” of the mechanotransduction channels, deceased fluorescence by only 44%. In contrast, P2X receptor antagonists (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid [PPADS], suramin, 2',3′-O-(2,4,6-trinitrophenyl) ATP [TNP-ATP], and d-tubocurarine) had greater effects on dye loading. PPADS, suramin, and TNP-ATP all decreased intracellular AM1-43 fluorescence in hair cells by at least 69% when applied at a concentration of 100 μM. The difference between d-tubocurarine-treated and control fluorescence was statistically insignificant when d-tubocurarine was applied at a concentration that blocks the mechanotransduction channel (200 μM). At a concentration that also blocks P2X2 receptors (2 mM), d-tubocurarine decreased dye loading by 72%. From these experiments, it appears that AM1-43 can enter hair cells through endogenously activated P2X receptors. Thus, the contribution of P2X receptors to dye entry should be considered when using styryl pyridinium dyes to detect hair cell mechanotransduction channel activity, especially in the absence of explicit mechanical stimulation of stereocilia.

Squalamine: An Appropriate Strategy against the Emergence of Multidrug Resistant Gram-Negative Bacteria?

We reported that squalamine is a membrane-active molecule that targets the membrane integrity as demonstrated by the ATP release and dye entry. In this context, its activity may depend on the membrane lipid composition. This molecule shows a preserved activity against bacterial pathogens presenting a noticeable multi-resistance phenotype against antibiotics such as polymyxin B. In this context and because of its structure, action and its relative insensitivity to efflux resistance mechanisms, we have demonstrated that squalamine appears as an alternate way to combat MDR pathogens and by pass the gap regarding the failure of new active antibacterial molecules.

Altered Traveling Wave Propagation and Reduced Endocochlear Potential Associated with Cochlear Dysplasia in the BETA2/NeuroD1 Null Mouse

The BETA2/NeuroD1 null mouse has cochlear dysplasia. Its cochlear duct is shorter than normal, there is a lack of spiral ganglion neurons, and there is hair cell disorganization. We measured vertical movements of the tectorial membrane at acoustic frequencies in excised cochleae in response to mechanical stimulation of the stapes using laser doppler vibrometry. While tuning curve sharpness was similar between wild-type, heterozygotes, and null mice in the base, null mutants had broader tuning in the apex. At both the base and the apex, null mice had less phase lag accumulation with increasing stimulus frequency than wild-type or heterozygote mice. In vivo studies demonstrated that the null mouse lacked distortion product otoacoustic emissions, and the cochlear microphonic and endocochlear potential were found to be severely reduced. Electrically evoked otoacoustic emissions could be elicited, although the amplitudes were lower than those of wild-type mice. Cochlear cross-sections revealed an incomplete partition malformation, with fenestrations within the modiolus that connected the cochlear turns. Outer hair cells from null mice demonstrated the normal pattern of prestin expression within their lateral walls and normal FM 1-43 dye entry. Overall, these data demonstrate that while tonotopicity can exist with cochlear dysplasia, traveling wave propagation is abnormally fast. Additionally, the presence of electrically evoked otoacoustic emissions suggests that outer hair cell reverse transduction is present, although the acoustic response is shaped by the alterations in cochlear mechanics.

Severe interference between retinal angiography and automated four-color flow cytometry analysis of blood mononuclear cells

Retinal angiography has become a widely used diagnostic tool. It requires the intravenous administration of the fluorescent dyes fluorescein and indocyanin green. We recently received blood taken 8 h after retinal angiography, without our knowing it. We describe the failure of an automated flow cytometry system in the enumeration of lymphocyte subpopulations in this sample. Cell enumeration was achieved by the use of the lyse-no wash MultiTEST procedure (Becton-Dickinson) together with the FACSCalibur cytometer. Absolute cell counts were obtained using TruCount beads. Data were analyzed automatically by the MultiSET and manually with the CellQuest softwares. The dot plots obtained with this sample looked quite abnormal. All monuclear cells stained brightly in the FITC channel irrespective of anti-CD3-FITC conjugate binding. This resulted in a major undercompensation for the increased spillover of the fluorescein emission into the PE-channel. PE-labeled cell and TruCount bead events coalesced. The MultiSET software failed to draw proper gatings and proved useless. Alternative manual gatings could partially rescue the analysis. Clinicians and cytometrists should be aware that, because of dye entry or binding, blood mononuclear cells collected shortly after retinal angiography are not suitable even for common cytometry applications.

Ocular Indigo Dye Injury

To describe the clinical course, management, and final outcome in a case of ocular injury caused by accidental fall of indigo dye in both eyes. Review of patient's record and relevant literature. A 32-year-old man sustained ocular injury caused by inadvertent indigo dye in both eyes. He presented with bluish staining of the entire ocular surface, subtotal corneal epithelial breakdown in both eyes, and entry of dye into the anterior chamber in the left eye. This was treated as acute alkali burns with lubricants and steroids. Over a period of 3 to 4 months, the ocular surface staining resolved completely, and visual acuity improved remarkably. Although the clinical presentation was initially a cause for concern, the condition resolved completely with the same conservative management as applicable for other chemical injuries.

Requirement for Annexin A1 in Plasma Membrane Repair

Ca2+ entering a cell through a torn or disrupted plasma membrane rapidly triggers a combination of homotypic and exocytotic membrane fusion events. These events serve to erect a reparative membrane patch and then anneal it to the defect site. Annexin A1 is a cytosolic protein that, when activated by micromolar Ca2+, binds to membrane phospholipids, promoting membrane aggregation and fusion. We demonstrate here that an annexin A1 function-blocking antibody, a small peptide competitor, and a dominant-negative annexin A1 mutant protein incapable of Ca2+ binding all inhibit resealing. Moreover, we show that, coincident with a resealing event, annexin A1 becomes concentrated at disruption sites. We propose that Ca2+ entering through a disruption locally induces annexin A1 binding to membranes, initiating emergency fusion events whenever and wherever required.

Does Prior Adjuvant Chemoradiotherapy Lead to a Safer Pancreatoduodenectomy?

The article by Cheng et al. is another attempt to compare the effects of preoperative versus postoperative adjuvant chemoradiotherapy (CRT) for presumably localized pancreatic adenocarcinoma by retrospective analysis of two nonrandomized treatment cohorts. Because the only significant differences in morbidity within the preoperative and postoperative cohorts in this study were in pancreatic leak and intra-abdominal abscess formation (favoring the preoperative CRT cohort), the primary focus of this study was the relative incidence of postoperative pancreatic leaks. The authors aver that preoperative CRT reduces the incidence of both pancreatic leaks and leak-associated morbidity and mortality, and they even go so far as to recommend preoperative CRT in those with ampullary cancer to decrease postoperative leaks. Is this information new, true, or relevant? Ishikawa et al. were the first to examine the question. They described two retrospective nonrandomized cohorts: one treated with preoperative radiotherapy (RT) and the other not. Patients with fistulas were defined as those with amylase levels >2000 U/L in their drains for >1 week. Furthermore, a fistula was defined as major if either bile was seen in the drain fluid or the fistulogram showed entry of dye into the jejunum (all patients had pancreatojejunostomy over external pancreatic stents). Minor fistulas were those without either of these findings but with the defined drain amylase levels. One of 22 patients with preoperative RT developed a major fistula, and 1 of 54 patients without preoperative RT had a major fistula; 3 of 54 had minor fistulas that became major, and 6 had minor fistulas. Because the two immediate major fistulas were attributed to premature withdrawal or occlusion of pancreatic stents, these cases were excluded. The 9 of 53 patients with minor or minor changing to major fistulas without RT were compared with the 0 of 21 patients without fistulas in the post-RT cohort: differences were significant (P = .05). Statistical significance here, however, required that the two major leaks be omitted. Clearly, those receiving preoperative RT seemed to do no worse than those without it. Lowy et al. examined pancreatic leak in patients with and without preoperative CRT in a randomized study of perioperative octreotide. Their definition of fistula was either biochemical (defined as increased drain amylase at postoperative day 3) or clinical (requiring reoperation or drainage). Whereas none of the 46 patients with protocol-based preoperative CRT for pancreatic cancer developed clinical pancreatic fistulas, there were enough with biochemical leaks that no difference was shown between those treated with preoperative CRT and those who had surgery first. Again, preoperative CRT seemed not to be a deterrent to safe recovery from pancreatoduodenectomy, but leaks were no less frequent than in those treated without preoperative therapy. Cheng et al. defined a leak as any drainage fluid at any time after surgery with a value three times that of serum amylase. Multivariate analysis showed that preoperative CRT produced a statistically significant decrease in pancreatic leaks (10.1% vs. 43.3%; P < .001). For those with pancreatic head cancer, 3 (4.8%) of 62 of those with preoperative CRT had a fistula, Received October 19, 2005; accepted October 26, 2005; published online January 1, 2006. Address correspondence and reprint requests to: John Hoffman, MD; E-mail: jp_hoffman@fccc.edu

Two phases of zymogen granule lifetime in mouse pancreas: ghost granules linger after exocytosis of contents

Different cell types show widely divergent mechanisms and kinetics of exocytosis. We investigated these processes in pancreatic acinar cells by using video-rate 2-photon microscopy to image entry of extracellular dye into individual zymogen granules undergoing exocytosis. Fluorescence signals display two distinct phases; an initial peak that then decays over several seconds to a prolonged plateau. Several observations suggest that the first component reflects the binding of dye to the granule contents and their subsequent release into the acinar duct. These observations include: the peak/plateau fluorescence ratio differs between different dyes; the initial fluorescence decay mirrors the loss of granule contents as monitored by differential interference contrast microscopy; and the fall in vesicular fluorescence is accompanied by a rise in fluorescence in the adjacent duct lumen. We thus propose the use of extracellular fluorescent probes as a convenient means to monitor the kinetics of loss of proteinaceous content from secretory granules. In pancreatic acinar cells the fusion pore remains open much longer than required to ensure secretion of the granule contents, and instead the persistent empty 'ghost-granule' may act as a conduit to which secondary granules can fuse and release their contents by compound exocytosis.

Sentinel lymph node mapping and selective lymphadenectomy: the standard of care for melanoma.

Selective sentinel lymphadenectomy (SSL) should be considered a standard of care approach for staging patients with primary invasive melanoma 1 mm or greater. It is essential that multidisciplinary teams should master the techniques of preoperative lymphoscintigraphy, intraoperative lymphatic mapping, and pathologic evaluation of the sentinel lymph nodes (SLNs). An SLN may be blue, hot, or any lymph node (LN) greater than 10% of the in-vivo count of the hottest LN. An enlarged or indurated LN should be removed because it may contain metastatic cancer cells that block blue dye or radiotracer entry. Frozen sections are not recommended. Surgeons who use isosulfan blue dye should be cognizant of treatment for a potentially fatal reaction. Prophylactic LN dissection should not be performed if a SSL can be performed as a staging procedure. A complete LN dissection is performed if the SLN is positive. It is important to follow the clinical outcome of patients undergoing SSL, thus its role can be further defined.

Lighting up the Senses: FM1-43 Loading of Sensory Cells through Nonselective Ion Channels

We describe a novel mechanism for vital fluorescent dye entry into sensory cells and neurons: permeation through ion channels. In addition to the slow conventional uptake of styryl dyes by endocytosis, small styryl dyes such as FM1-43 rapidly and specifically label hair cells in the inner ear by entering through open mechanotransduction channels. This labeling can be blocked by pharmacological or mechanical closing of the channels. This phenomenon is not limited to hair cell transduction channels, because human embryonic kidney 293T cells expressing the vanilloid receptor (TRPV1) or a purinergic receptor (P2X2) rapidly take up FM1-43 when those receptor channels are opened and not when they are pharmacologically blocked. This channel permeation mechanism can also be used to label many sensory cell types in vivo. A single subcutaneous injection of FM1-43 (3 mg/kg body weight) in mice brightly labels hair cells, Merkel cells, muscle spindles, taste buds, enteric neurons, and primary sensory neurons within the cranial and dorsal root ganglia, persisting for several weeks. The pattern of labeling is specific; nonsensory cells and neurons remain unlabeled. The labeling of the sensory neurons requires dye entry through the sensory terminal, consistent with permeation through the sensory channels. This suggests that organic cationic dyes are able to pass through a number of different sensory channels. The bright and specific labeling with styryl dyes provides a novel way to study sensory cells and neurons in vivo and in vitro, and it offers new opportunities for visually assaying sensory channel function.

Blockade of maitotoxin-induced endothelial cell lysis by glycine and L-alanine.

The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs) is a model for oncotic/necrotic cell death. The cascade is initiated by an increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)), which is followed by the biphasic uptake of vital dyes. The initial phase of dye entry reflects activation of large pores and correlates with surface membrane bleb formation; the second phase reflects cell lysis. In the present study, the effect of the cytoprotective amino acid glycine was examined. Glycine had no effect on MTX-induced change in [Ca(2+)](i) or on the first phase of vital dye uptake but produced a concentration-dependent (EC(50) approximately 1 mM) inhibition of the second phase of dye uptake. No cytoprotective effect was observed with l-valine, l-proline, or d-alanine, whereas l-alanine was equieffective to glycine. Furthermore, glycine had no effect on MTX-induced bleb formation. To test the hypothesis that glycine specifically blocks formation of a lytic "pore," the loss of fluorescence from BAECs transiently expressing GFP and concatemers of GFP ranging in size from 27 to 162 kDa was examined using time-lapse videomicroscopy. MTX-induced loss of GFP was rapid, correlated with the second phase of dye uptake, and was relatively independent of molecular size. The MTX-induced loss of GFP from BAECs was completely blocked by glycine. The data suggest that the second "lytic" phase of MTX-induced endothelial cell death reflects formation of a novel permeability pathway that allows macromolecules such as GFP or LDH to escape, yet can be prevented by the cytoprotective agents glycine and l-alanine.

FM1-43 Dye Behaves as a Permeant Blocker of the Hair-Cell Mechanotransducer Channel

Hair cells in mouse cochlear cultures are selectively labeled by brief exposure to FM1-43, a styryl dye used to study endocytosis and exocytosis. Real-time confocal microscopy indicates that dye entry is rapid and via the apical surface. Cooling to 4 degrees C and high extracellular calcium both reduce dye loading. Pretreatment with EGTA, a condition that breaks tip links and prevents mechanotransducer channel gating, abolishes subsequent dye loading in the presence of calcium. Dye loading recovers after calcium chelation with a time course similar to that described for tip-link regeneration. Myo7a mutant hair cells, which can transduce but have all mechanotransducer channels normally closed at rest, do not label with FM1-43 unless the bundles are stimulated by large excitatory stimuli. Extracellular perfusion of FM1-43 reversibly blocks mechanotransduction with half-blocking concentrations in the low micromolar range. The block is reduced by high extracellular calcium and is voltage dependent, decreasing at extreme positive and negative potentials, indicating that FM1-43 behaves as a permeant blocker of the mechanotransducer channel. The time course for the relief of block after voltage steps to extreme potentials further suggests that FM1-43 competes with other cations for binding sites within the pore of the channel. FM1-43 does not block the transducer channel from the intracellular side at concentrations that would cause complete block when applied extracellularly. Calcium chelation and FM1-43 both reduce the ototoxic effects of the aminoglycoside antibiotic neomycin sulfate, suggesting that FM1-43 and aminoglycosides enter hair cells via the same pathway.

Translocon Pores in the Endoplasmic Reticulum Are Permeable to a Neutral, Polar Molecule

The pore of the translocon complex in the endoplasmic reticulum (ER) is large enough to be permeated by small molecules, but it is generally believed that permeation is prevented by a barrier at the luminal end of the pore. We tested the hypothesis that 4-methylumbelliferyl alpha-d-glucopyranoside (4MalphaG), a small, neutral dye molecule, cannot permeate an empty translocon pore by measuring its activation by an ER resident alpha-glucosidase, which is dependent on entry into the ER. The basal entry of dye into the ER of broken Chinese hamster ovary-S cells was remarkably high, and it was increased by the addition of puromycin, which purges translocon pores of nascent polypeptides, creating additional empty pores. The basal and puromycin-dependent entries of 4MalphaG were mediated by a common, salt-sensitive pathway that was partially blocked by spermine. A similar activation of 4MalphaG was observed in nystatin-perforated cells, indicating that the entry of 4MalphaG into the ER did not result simply from the loss of cytosolic factors in broken cells. We reject the hypothesis and conclude that a small, neutral molecule can permeate the empty pore of a translocon complex, and we propose that translationally inactive, ribosome-bound translocons could provide a pathway for small molecules to cross the ER membrane.

Effect of eptifibatide on coronary flow reserve following coronary stent implantation (An ESPRIT substudy)

P coronary intervention with stenting improves epicardial large vessel lumen diameters but can be detrimental to the microcirculation as a result of either downstream embolization or vasospasm. Coronary flow reserve (CFR) is a measurement of the capacity of blood flow that is augmented in response to adenosine (hyperemic flow) and is a valuable tool in assessing the integrity of the microvasculature after stent placement. We hypothesized that patients treated with eptifibatide in the Enhanced Suppression of the Platelet IIb/IIIa Receptor with Integrilin Therapy (ESPRIT) trial1 would have improved CFR after elective stent placement compared with patients receiving placebo. Furthermore, we hypothesized that tissue level perfusion would be improved with eptifibatide therapy, and we assessed the kinetics of myocardial perfusion using digital subtraction angiography. • • • The ESPRIT trial was a double-blind, multicenter, randomized, parallel group, placebo-controlled trial of planned, nonemergency stenting of native coronary arteries in 2,064 patients.1 Patients were allocated in a 1:1 ratio between eptifibatide and placebo immediately before planned percutaneous coronary stent implantation. Eptifibatide was administered as a 180 mg/kg bolus followed by a 2.0 mg/kg/min infusion for 18 to 24 hours, with a second 180 mg/kg bolus given 10 minutes after the first. An angiographic substudy was conducted at 3 sites that enrolled 65 patients to assess CFR and myocardial perfusion at the completion of the intervention. The Corrected Thrombolysis In Myocardial Infarction (TIMI) Frame Count (CTFC), the number of cine frames required for contrast to first reach standardized distal coronary landmarks in the culprit artery, was measured using a frame counter on a cine viewer.2,3 After stenting, patients received intracoronary adenosine (24 to 36 mg in the left anterior descending artery and left circumflex artery; 18 to 24 mg in the right coronary artery). The CTFC was assessed after stenting and 15 seconds after adenosine administration in the primary culprit lesion by the angiographic core laboratory, which was blinded to treatment assignment. CFR was calculated as the ratio of preadenosine CTFC divided by postadenosine CTFC, which has been validated in the literature as highly correlated with Doppler-derived CFR (r 5 0.88, p ,0.0001).4 To quantitate the kinetics of dye entry into the myocardium, digital subtraction angiography was used. Digital subtraction angiography was performed at end-diastole by aligning cine frame images before dye filled the myocardium with the frame in which dye first reached its peak brightness. The spine, ribs, diaphragm, and the epicardial artery were then subtracted. A representative region of the myocardium was sampled that was free of overlap by epicardial arterial branches to determine the increase in the grayscale brightness of the myocardium. The circumference of the myocardial blush was measured using a handheld planimeter (Fowler, Inc., Medford, Massachusetts). The frame count divided by the number of frames per second was used to measure the time elapsed during angiography to quantitate the rate of increase in the growth (centimeters per second) and brightness (gray per second) of myocardial blush. The digital subtraction angiographic reserve was calculated as the relative improvement in the rate of increase in brightness after adenosine: after adenosine (gray per second) divided by after stenting (gray per second). Blush was also assessed visually using the TIMI myocardial perfusion grade.5 The size (centimeters) and brightness of the myocardium (gray) were multiplied together to yield gray centimeters per second as a simultaneous index, integrating brightness and size of myocardial perfusion. Analyses were performed using Stata statistical software version 6.0.6 Variables were compared using the Fisher’s exact test or chi-square test for categorical data and the Student’s t test for continuous variables. Variables known to impact the CTFC (culprit location and reference diameter) and myocardial blush were adjusted for in multivariate models.5,7 There was no difference between patients who received placebo and those on eptifibatide with respect to baseline and postintervention angiographic characteristics (Table 1). CFR was greater among patients who received eptifibatide than patients on placebo (1.78 6 0.95, n 5 16 vs 1.28 6 0.40, n 5 27; p 5 From the Cardiovascular Division, Department of Medicine, the University of California San Francisco, San Francisco, California; The Beth Israel Deaconess Medical Center, Boston, Massachusetts; Sunnybrook Health Science Center, Toronto, Ontario, Canada; Jackson/ Madison County General Hospital, Jackson, Tennessee; COR Therapeutics, San Francisco, California; and Duke Clinical Research Institute, Durham, North Carolina. This study was supported in part by a grant from Cor Therapeutics, Inc., South San Francisco, California. Dr. Gibson’s address is: Chief of Interventional Cardiology, University of California San Francisco, 3333 California Street, Suite 430, San Francisco, California 94118. E-mail: mgibson@perfuse.org. Manuscript received October 25, 2000; revised manuscript received and accepted January 5, 2001.

Expression of ryanodine receptor RyR3 produces Ca2+ sparks in dyspedic myotubes.

Discrete, localized elevations of myoplasmic [Ca2+], Ca2+ 'sparks', were readily detected using the fluorescent Ca2+ indicator fluo-3 and laser scanning confocal microscopy in 'dyspedic' 1B5 myotubes, i.e. myotubes which do not express ryanodine receptors (RyRs), transduced with virions containing cDNA for RyR type 3 that were saponin permeabilized to allow dye entry. Ca2+ sparks were never observed in non-transduced RyR null myotubes. The spatial locations of sparks observed in permeabilized myotubes roughly corresponded to regions of RyR protein expression in the same myotube as detected after subsequent fixation and antibody staining. Permeabilized RyR3-transduced myotubes exhibited similar punctate peripheral RyR3 protein immunohistochemical patterns as myotubes fixed before permeabilization indicating that permeabilization did not affect the structural organization of the triad. Ca2+ sparks, recorded in line scan mode, in permeabilized myotubes expressing RyR3 exhibited mean amplitudes (change in fluorescence/mean fluorescence, DeltaF/F: 1.20 +/- 0.04) and temporal rise times (10-90%; 6.31 +/- 0.12 ms) similar to those of sparks recorded in permeabilized frog skeletal muscle fibres (0.98 +/- 0.01; 6.11 +/- 0.07, respectively) using the same confocal system. Spatial extent and temporal duration of the Ca2+ sparks were approximately 40% larger in the RyR3-expressing myotube cultures than in frog fibres. Ca2+ sparks recorded in line scan mode often occurred repetitively at the same spatial location in RyR3-expressing myotubes. Such repetitive events were highly reproducible in amplitude and spatio-temporal properties, as previously observed for repetitive mode sparks in frog skeletal muscle. Ca2+ sparks recorded in xy mode were frequently compressed in the y (slower scan) direction compared to the x direction. This asymmetry was reproduced assuming spatially symmetric events having the time course of Ca2+ sparks recorded in line scan (xt) mode. These expression studies demonstrate that the presence of RyR3 is sufficient for the production of Ca2+ sparks in a skeletal muscle system lacking the expression of any other RyR isoform.